Characterisation of deletion mutants in the hydrophobic core of the prion protein . 111

In 1998, Hölscher and co-workers reported that the deletion mutant PrPΔ114-121 (114Δ8) possessed a dominant negative effect: the mutant was not convertible into PrP^Sc and prevented conversion of endogenous PrP^C into PrP^Sc [172]. In order to determine the minimal deletion size that would lead to a significant inhibition of conversion smaller mutations in this region (PrP∆114-115 (114Δ2), PrP∆114-117 (114Δ4), PrP∆114-119 (114Δ6), PrP∆116-119 (116Δ4), PrP∆116-121 (116Δ6) and PrP∆118-121 (118Δ4) had been created to investigate their conversion into PrP^Sc in a scrapie infected cell culture system.

The convsersion efficiency of the deletion mutants was monitored with IF and WB assays.

With IF, mutant PrP^Sc was detected only after transduction of PrP-wt and 114Δ2 (Figure 41). The final proof for convertibility was obtained by WB analysis. PrP-wt and 114Δ2 were the only prion protein constructs which could be converted into PrP^Sc (Figure 43).

Table 1 summarises the results of the conversion study and narrows down the region of amino acids necessary for conversion. Included is the deletion mutant of the palindromic sequence 112-AGAAAGA-119 (112Δ7) within the hydrophobic domain (aa 111-134) of the prion protein known to be dominant negative in the literature [173].

The deletion mutant 114Δ2 showed no sign of conversion into PrP^Sc. Of note, the deletion construct 114Δ2 could also be called 116Δ2. Both shared the identical sequence. From the 4 amino acids 114-AAAA-117 two adjecent alanines have to be deleted to be either defined as 114Δ2 or 116Δ2. Therefore, one can assume that 116Δ2 is convertible into PrP^Sc. Remarkably the mutant 114Δ4 was not convertible into PrP^Sc although all 4 alanines were deleted. It seemed that deletion of at least 4 amino acids was a prerequisite for inhibition of conversion. Thereby, it was not necessary which amino acids in the hydrophobic region were deleted as 116Δ4 and 118Δ4 also showed no sign of conversion.

For future experiments it will be interesting to investigate the behaviour of other mutants with only 2 or 3 amino acids deleted. Deletion mutants like 118Δ2, 120Δ2, 113Δ3 and 116Δ3 could be examined for their ability to convert into PrP^Sc. Convertibility of mutants with only 2 deleted residues will strengthen the finding that two amino acids are not

Discussion 112

sufficient to induce conversion. With deletion of 3 amino acids within the TM1 region it will be either determined that even 3 amino acids are sufficient for inhibition of conversion or to confirm the result that at least 4 amino acids within the TM1 region are required for conversion.

Table 1: Results of conversion study from deletion mutants within the hydrophobic domain (aa 111-134) of the prion protein.

Deletion mutant Deleted amino acids Conversion Source 112Δ7 112 A G A A A A G A 119 no [173]

Chabry and collegues argued that the presence of residues 119 and 120 were crucial for an efficient inhibitory effect [170]. Peptides from the region 119-141 inhibited PrP^Sc formation while peptide 121-141 did not. This finding cannot be supported by the present study. Amino acids 119-AV-120 seemed not to be crucial for PrP^Sc formation since even 114Δ4 showed an inhibition of conversion. It is not confirmed that only the hydrophobic core is essential for prion conversion. Peptides from aa 119-136, 166-179 and 200-223 inhibited PrP^Sc formation in vitro and in cell culture [248]. Upon binding of the peptide to PrP^C the interaction of PrP^C with PrP^Sc was inhibited and conversion was prevented. The mutant Q218K was also reported to inhibit conversion [249]. Thus, not only residues from aa 111-134 of the hydrophobic region were critically involved in the intermolecular interactions that lead to PrP^Sc formation but also other sites of the prion protein. These findings supported the hypothesis that structural alterations in the prion protein due to mutations lead to inhibition of conversion [249, 250]. Due to structural alterations in the molecule PrP^C is no longer able to bind to PrP^Sc.

In the present study it was demonstrated that only 4 amino acids in the TM1 region were sufficient to abrogate conversion. A structural change compared to PrP-wt may be the reason in this case as proposed above. The deletion mutant 114Δ8 which exerts a dominant negative effect did not show an overall altered structure compared to wt but it showed an additional short β-sheet which stabilised 114Δ8. That could lead to a higher energy barrier at interaction with PrP^Sc which made it impossible for 114Δ8 to convert into PrP^Sc [174]. Conceivable is also that deletion of eight (114Δ8) or even four (this work) amino acids result in a tight binding to PrP^Sc with higher affinity than wt, because endogenous PrP-wt could not displace 114Δ8 from PrP^Sc and could not be converted [172]. Probably, even overexpression of the mutant protein could be sufficient to prevent binding of endogenous PrP-wt to PrP^Sc by displacing.

Interaction between PrP^C and PrP^Sc could either take place directly or indirectly mediated through another receptor, called protein X. Protein X is postulated to play a role in the conversion process of another dominant negative mutant Q218K [189, 249, 251, 252].

The mutant Q218K binds to protein X with a higher affinity than wt and then this complex is removed from the replication process. Several interaction partners could be identified which bind to PrP^C, some within the hydrophobic domain: residues 110-128 of PrP^C is the binding site for heparan sulfate proteoglycans [253], aa 113-128 for stress-inducible protein 1 (STI-1) [63] and aa 144-179 for LRP/LR (laminin receptor precursor/laminin receptor) [198]. While the roles of LRP/LR and glucosaminoglycans remain to be established, STI-1 interaction with PrP^C reveals a function in neuroprotection possibly by activation of SOD activity [254, 255] and in neuritogenesis [64, 256]. Thus, the conversion site may overlap with functional sites of PrP^C.

6.2.2 Experimental setup for investigation of the convertibility of deletion mutants In the first step, transient transfections with deletion mutants were carried out in H6-22L cells but the transfection efficiency was too low to detect appropriate protein signals in WBs (not shown). Therefore, the experimental approach changed to lentiviral transduction. Over 70 % transduced cells could be obtained in scrapie infected cells with this method (Figure 38) while in transient transfections the efficiency was only about 30-40 %. Because lentiviral vectors are able to transduce post-mitotic cells such as terminally differentiated neurons, this technique offered a tool to investigate the phenotype of PrP

Discussion 114

mutations in such cells [189]. Preliminary experiments showed that the experimental setup for a putative conversion of deletion mutants worked well. PrP-wt was convertible into PrP^Sc and acted as a positive control while 114Δ8 was not convertible and acted as a negative control (Figure 42).

For transduction of scrapie infected H6-22L cells a MOI of 15 was chosen since with this MOI the best transduction efficiency (about 70 %) could be achieved (Figure 38). As expected, the transduction efficiency with a special MOI changed highly from one experiment to another. For example, with a MOI of 20 the efficiency varied from 44 % to 87 %. For determination of the minimal deletion size necessary for conversion these observations had to be considered. Therefore, a calibration was performed. This was done by determination of the signal intensities when equal protein amounts of non PK treated samples were loaded on the SDS-PAGE (Figure 39). The signal intensities gave a hint for the transduction efficiencies. Due to these signal intensities equal transduction efficiencies were calculated. Thus, in Figure 42 not equal protein amounts were loaded but equal transduction efficiencies.

It seemed as if the conversion efficiency for 114Δ2 and the positive control PrP-wt was identical but one had to be careful with this conclusion. In three independent experiments the signal intensity was not always the same for 114Δ2 and PrP-wt although a calibration for loading of equal transduction efficiencies was performed. Thus, only a qualitative rather than a quantitative conclusion could be drawn. In each experiment either IF or WB, a PrP^Sc signal was present for 114Δ2 indicating that only 114Δ2 is convertible into PrP^Sc while all other mutants are resistant to conversion.